Nature and Science 2010;8(10)

Development of vegetative and sexual multiplication protocol for commercialization of racemosa Hook. f. – a critically endangered medicinal of N.W. Himalaya

Peerzada Arshid Shabir*, Irshad Ahmad Nawchoo** and Aijaz Ahmad Wani Economic Botany and Reproductive Biology Laboratory, Department of Botany,University of Kashmir, Srinagar-190 006. J&K, India *email:[email protected] **email: [email protected]

Abstract: Inula racemosa is an important and critically endangered medicinal plant with tremendous potential as an important natural resource. The development of conservation and commercialization technique is a priority at present. To assess this, rhizome splitting as a means of vegetative propagation and seed germination for sexual propagation were evaluated for mass multiplication of this potent medicinal herb of North Western . Split rhizome cuttings treated with varying concentrations of IAA, IBA and GA3 showed 88.89±0.95% sprouting and 77.78±1.42% of rooting in 100ppm of IAA. The studies undertaken on the seed germination of I. racemosa as a means of mass multiplication revealed that seeds show a broad range of pre-chilling requirements. Highest germination percentage- 90.00±0.30% were recorded when scarification and GA3 (100ppm) were applied together. Mean germination time declined with higher concentrations of GA3 applied to scarified seeds and also with increased duration of stratification. [Nature and Science 2010;8(10):246‐252]. (ISSN: 1545‐0740).

Key Words: Inula racemosa, vegetative propagation, germination, scarification, stratification, mass multiplication.

1. Introduction sitisterol, daucosterol, inunolide, aplotexene, North West Himalayas, one of the richest phenylacetonitrite and isoinunal (Kalsi et al., 1989, pools of biological diversity in the World, has been and Wang et al., 2000). Allantolactone and experiencing ruthless extraction of wild medicinal isoalantolactone are the major constituents known to due to ever increasing global inclination possess antifungal properties (Satyawati et al., towards use of herbal medicine and thus 1987). The diverse active ingredients of the Inula endangering many of its high value gene stock. The root are being utilized as therapeutic components continued commercial over-exploitation of these since time immemorial in both organized (Ayurveda, valuable herbal gems has resulted in depleting the Unani) and unorganized (folk, tribal, native) form. population of many such species in their natural The roots of I. racemosa have been used as folk habitats. In India, with an estimated 8000 species of medicine in East Asia and Europe (Kuda, 1986). medicinal plants (Joy et al., 1998), more than 90% Native Americans used this plant for treatment of of medicinal plants for herbal drug industries are tuberculosis (Moerman, 1986). The alantolactone drawn from natural habitats, thereby putting them to obtained from roots of I. racemosa enhances insulin severe exploitation (Gupta et al., 1998, and Ved et sensitivity and thus proves beneficial in fat reduction al., 1998). In addition to the over exploitation, (Tripathi and Chaturvedi, 1995). Roots of I. destructive harvesting practices, habitat degradation racemosa are also useful in treating pulmonary and and agricultural encroachment to wild habitats have cardiovascular disorders (Miller, 1998, and also been recognized as contributing factors in the Lokhande et al., 2006) loss of our herbal gems. Due to the great market demand, over and Inula racemosa Hook. f., a critically endangered illegal exploitation from wild, and the endangered himalayan herb (Anonymous, 1998), commonly status of the species, the present study was carried known as “Pushkarmoola” of family , out to ensure proper utilization, commercialization was targeted for the present study. Distributed from as well as conservation of the species. The plant temperate to alpine belts of Kashmir and Himachal propagation is generally regarded as the first phase Pradesh, this perennial herb, known for its potent of the complete package of productive technology of medicinal properties, is facing ruthless over- herbal medicine. The vegetative and sexual exploitation and is at the verge of extinction. The propagation have been recognized as efficient means chemical profiling of plant roots have shown the of rapid multiplication and conservation of presence of alantolactones and isoalontolactones, important taxa. Keeping in view all the above facts,

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the present study was carried out to develop a mass where ‘n’ is number of newly germinated seeds multiplication protocol for conservation and after each incubation period in days ‘d’, and ‘N’ is commercialization of Inula racemosa and the total number of seeds germinated at the end of information will also prove beneficial for the other experiment. The seed vigour index was related species. calculated using the formula of Abdul-baki and Anderson (1973) i.e. Material and Methods: VI=Ls×Pg/100 Rhizome was used as the main means of where, ‘Ls’ is the mean of seedling length and ‘Pg’ vegetative propagation with an emphasis on its is percentage germination. economic potential. In the month of April 2009, rhizomes of I. racemosa were collected from its Data Analysis: natural habitat ‘Drung Farozpur’ at an altitude of The data was analysed statistically using 2150m (amsl). The freshly harvested rhizomes were MS-Excel 2007. Data was analysed for Mean and washed thoroughly with running tap water followed standard deviation. ANOVA was used to interpret by double distilled water to run off any contaminant. the variation and to identify the best treatments. Each rhizome portion was split longitudinally with a sterilized razor blade/knife into 2, 4 or 8 pieces, Results and Discussion: according to the size of the parental rhizome, with Results of rhizome splitting as a means of each piece having at least one vegetative bud. The mass multiplication for I. racemosa are shown in split rhizome cuttings were treated with different Table 1. Results indicate a maximum shoot concentrations of hormonal viz. IAA, IBA and GA3 sprouting and percentage survival (rooting) in (25ppm, 50ppm and 100ppm) for 48hours. One set 100ppm of IAA (88.89% and 77.78% respectively), in each case was treated with distilled water to serve as compared to control with 77.78% of sprouting as control. In each treatment three replicates with six and 44.45% of rooting. Both IAA as well as IBA cuttings each were used and subsequently sown in were found to have a profound effect on sprouting as sandy loam textured soil in the Kashmir University well as rooting percentage. Similar results have been Botanical Garden (KUBG), Srinagar, India. reported by Butola and Badola, (2007) in Angelica Percentage sprouting and rooting was recorded for glauca and Heracleum candicans and by Vashistha each treatment. et al., (2009) in Angelica glauca and Angelica Fresh seeds used for the study undertaken archangelica. Among different treatments were collected from one population in natural IAA(100ppm) and IBA(100ppm) proved to be most habitats of I. racemosa in Oct. 2009 and were stored effective (p≤0.05) in increasing the sprouting and for two months at room temperature. The air dried rooting percentage. GA3 concentrations were found seeds were surface sterilized by dipping in 0.01% to be less effective particularly in the induction of aqueous solution of mercuric chloride (Hgcl2) for 10 adventitious root development. to 15 seconds to discourage fungal infection Increased percentage of rooting after IAA followed by washing 2-3 times with double distilled and IBA treatments as compared to control shows water. The disinfected seeds were placed in the higher potential of IAA and IBA in the induction sterilized petriplates lined with a single layer of of adventitious root development as also reported whattman filter paper and subjected to different earlier by Kuris et al., (1980), in Origanum vulgare physical and chemical treatments. Three replicates L. Moreover, the chances of survival and growth of seeds were used for each treatment and the performance of vegetatively propagated individuals control sets were maintained in each case using are better implying that a reasonable number of double distilled water. The germination count was plantlets could be raised and their survival could be made daily wherein the emergence of the radical ensured by using different concentrations of IAA was used as the criterion to determine germination. and IBA. Thus the IAA and IBA in concentrations Mean germination time was calculated following ranging from 50-100ppm can be recommended as Hartman and Kester (1989) i.e.: promising treatments while carrying out vegetative MGT= ∑ (nd)/N mass multiplication of I . racemosa .

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Table: 1 Effect of Growth hormones on vegetative propagation of I. racemosa using rhizome segments. S. No Treatments Sprouting percentage Rooting percentage 1. Control 77.78±1.25 44.45±1.69 2. IAA 25ppm 72.23±0.95 61.12±0.48 3. IAA 50ppm 77.78±0.48 66.67±0.82 4. IAA100ppm 88.89±0.95* 77.78±1.42* 5. IBA 25ppm 61.12±0.95 50.00±0.82 6. IBA 50ppm 66.67±0.82 55.56±0.95 7. IBA 100ppm 83.34±1.42 61.12±0.48 8. GA3 25ppm 50.00±0.82 33.34±1.42 9. GA3 50ppm 66.67±1.42 50.00±1.64 10. GA3 100ppm 55.56±0.48 33.34±0.82 LSD(p≤0.05) 16.37 18.61 *=significant

Figure 1. Parental rhizome used for Figure 2. Splitted rhizome cuttings wherein Vegetative propagation each portion has part of apical meristem

Figure 3. One Rhizome segment showing Figure 4. A progenial mature individual shoot regeneration

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Figure 5. Graph showing effect of different growth hormones on sprouting and survival of vegetative cuttings of I. racemosa.

The effects of different pretreatments on I. also unable to induce germination in I. racemosa racemosa seed germination are shown in Table 2. seeds which seems to be controvertial with the The results showed significant (p≤ 0.05) differences results obtained by Sharma et al., (2006). among method used for stimulation of I. racemosa Scarification alone gave a good germination seed germination and no germination was recorded percentage but scarification and GA3 together on fresh seeds prior to onset of scarifaction or proved more effective and highest germination stratification. Among the pretreatments used, T10 percentage were obtained when GA3 in higher (scarification + GA3 100ppm) recorded the highest concentations were accompanied with scarification. germination of 90.00±0.30% followed by T21 (wet Decline in mean germination time (MGT) upto stratification of 100 days) with 86.67± 0.34%. 2.92±0.23 (Table 2 ) and highest value of seed Germination was absent in control replicates. Seeds vigour index 2.74±0.27 was also observed when showed a broad range of prechilling requirement for GA3 (100ppm) and scarification were applied germination. Dry and wet stratification for 40, 60, together. Appreciable reduction in MGT with 80, and 100 days increased the percentage increased concentrations of GA3 were also reported germination from 46.67±0.49% (dry stratification by Pradhan et al., (2010) in Swertia chirayita. GA3 for 40 days) to 86.67±0.34% (wet stratification for has been reported earlier also to mediate seed 100 days). The stratification treatments for I. germination by inducing the production of racemosa seed germination were also reported by hydrolytic enzymes in barley (Mozer, 1980). Other Sharma et al., (2006) and Jabeen et al., (2007). The traits such as radicle length, plumule length, increased time of statification increased total seed germination rate and vigour index were also affected germination and reduced mean germination time. by GA3 concentrations (Table 2). MGT in I. Ren and Tao, (2004) also reported that cold racemosa seeds decreased in all effective stratification treatments significantly promoted pretreatments compared to control where overall germination in Calligonium species. The germination is absent. Among the observed treatments T1-T6,T11-T13, and T22 proved to be effective pretreatments, T10 shows the significant ineffective and failed to stimulate the germination of reduction in MGT (2.92±0.23 days). MGT also I. racemosa. In comparision to mechanical decreased with increasing duration of stratification. scarification (removal of seed coat), acid Similar results were reported by Bhatt et al., (2000) scarification (Dip in H2SO4) was found ineffective in in Myrica esculanta. stimulating seed germination and GA3 alone was

Table II. Effect of different pretreatments on I. racemosa seed germination. S Treatment Days for first Percentage MGT Redicle Plumule Seed NO. germination germination length length vigour (cm) (cm) index T0 Control × × × × × × T1 GA3 × × × × × ×

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T2 Thiourea × × × × × × T3 IAA × × × × × × T4 IBA × × × × × × T5 Dip in × × × × × × H2SO4 T6 KNO3 × × × × × × T7 Scarification 4.66±0.95 70.00 ±0.45 4.96±0.69 0.78±0.08 0.72±0.08 1.05±0.19 (mechanical) T8 Scarification 5.68±0.48 63.34±0.49 4.49±0.83 0.85±0.06 0.97±0.07 1.15±0.03 +GA3 25ppm T9 Scarification 4.35±0.48 76.67±0.42 3.57±0.29 1.1±0.09 0.99±0.08 1.59±0.14 +GA3 50ppm T10 Scarification+ 1.35±0.47* 90.00±0.30* 2.92±0.23* 1.47±0.69* 1.56±0.09* 2.74±0.27 GA3 100ppm * T11 Kinetin × × × × × × T12 Dry × × × × × × stratification (20 days) T13 Wet × × × × × × stratification (20 days) T14 Dry 8.34±0.48 46.67±0.49 5.31±0.14 0.73±0.08 0.68±0.06 0.66±0.09 stratification (40 days) T15 Wet 7.67±0.29 53.34±0.49 4.89±0.27 0.82±0.09 0.83±0.07 0.89±0.11 stratification (40 days) T16 Dry 6.00±0.82 50.00±0.50 5.03±0.27 0.73±0.91 0.76±0.06 0.74±0.09 stratification (60 days) T17 Wet 6.34±2.13 46.67±0.48 4.86±0.46 0.88±0.90 0.89±0.06 1.06±0.12 stratification (60 days) T18 Dry 5.34±0.39 73.34±0.44 3.69±0.26 0.81±0.05 0.88±0.09 1.23±0.08 stratification (80 days) T19 Wet 4.34 ±0.48 80.00±0.40 3.57±0.11 0.75 ±0.03 0.86 ±0.08 1.29±0.13 stratification (80 days) T20 Dry 4.00 ±0.82 76.67 ±0.42 3.91±0.21 0.95±0.09 0.92 ±0.08 1.46±0.21 stratification (100 days) T21 Wet 3.67 ±1.74 86.67 ±0.34 3.81±0.52 1.01 ±0.16 0.99 ±0.16 1.61±0.12 stratification (100 days) T22 Dark × × × × × × T23 Dark+ 4.34 ±1.25 60.00 ±0.49 4.74±0.51 0.99 ±0.09 1.13 ±0.09 0.25±0.29 scarification LSD ≤0.05 1.51 *Significant

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Figure 6. Graph showing percentage germination in different effective treatments, wherein T7–T23 represents- Scarification, Scarification+GA3 25ppm, scarification+GA3 50ppm, Scarification+GA3 100ppm, Dry Stratification 40 days, Wet stratification 40 days, Dry stratification 60 days, Wet stratification 60 days, Dry stratification 80 days, Wet Stratification 80 days, Dry Stratification 100 days, wet stratification 100 days and Dark+scarification.

Figure 7. Germination of seeds after 100 days of wet stratification.

Germination percentage of scarified seeds of I. boosting rural economy and the information will prove racemosa were higher in light (70.00±0.45) than in beneficial for other related species. darkness(60.00±0.49). Gulzar et al., (2007) also recorded an enhanced germination percentage in light compared to Acknowledgement: germination in dark in Desmostachya bipinnata. The first author is grateful to Council of Conclusion: Plant propagation multiplies plants in bulk Scientific and Industrial Research, (CSIR) for providing and preserves their essential genetic charecteristics. The financial assistance as JRF. outcome of the present study can be gainfully utilized for multiplication of the species.Individuals raised through References: vegetative or sexual propagation could be utilized for 1. Abdul-baki AA and Anderson JD. Vigor determination revegetating the wild population and sustainable in Soybean seed by multiplication.Crop Sci. 1973; 3: 630- utilization of the species.The information will prove 633. beneficial not only for conservation of species but also in 2.Anonymous. Threatened Medicinal plants of Himalaya- A check list of CAMP Workshop, Lucknow.1998

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